The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Chengchao Zheng - One of the best experts on this subject based on the ideXlab platform.
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tm6 a novel nuclear Matrix Attachment Region enhances its flanking gene expression through influencing their chromatin structure
Molecules and Cells, 2013Co-Authors: Jiedao Zhang, Guodong Yang, Jinguang Huang, Chengchao ZhengAbstract:Nuclear Matrix Attachment Regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter Regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 Region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) Region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.
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functional characterization of a tobacco Matrix Attachment Region mediated enhancement of transgene expression
Transgenic Research, 2009Co-Authors: Jiedao Zhang, Guodong Yang, Chengchao ZhengAbstract:TM2, a new Matrix Attachment Region (MAR) isolated from tobacco, increases transgene expression in plants. We have carried out a more detailed analysis of the DNA elements in TM2 with the aim of improving its effect on transcription activation. Our study of the location effect of individual MARs on the expression of the adjacent 35S:gusA cassette indicated that the TM2 functions in a bidirectional manner, with the 5′-MAR being more efficient in enhancing β-glucuronidase expression than the 3′-MAR. The influence of 5′-MAR on different linked mini-promoters in transgenic tobacco cells suggested that the role of TM2 depends on the basic expression of the transgenes. Deletion analysis of one topo II site and two unwinding sites together with one T-box revealed that all these sites contribute most (93.3%) of the transcription activation mediated from the TM2 sequence. Additionally, micrococcal nuclease accessibility of the 35S promoter Region can be strengthened by linked TM2, suggesting that the TM2 mediates the spreading of nucleosome opening. Taken together, our results reveal that the TM2 mediates a more open and accessible chromatin DNA structure for promoter-dependent active transcription, which in turn enhances transgene expression.
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high transformation frequency of tobacco and rice via agrobacterium mediated gene transfer by flanking a tobacco Matrix Attachment Region
Physiologia Plantarum, 2007Co-Authors: Mengmeng Zhang, Yutao Yang, Hua Xue, Chengchao ZhengAbstract:Nuclear Matrix Attachment Regions (MARs) are suggested to regulate chromatin structure and influence the expression of flanking genes. Our previous study showed that TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear Matrix in vitro and increase transgene expression in vivo. Here, we investigated the role of TM2 MAR in improving transformation frequency of Agrobacterium-mediated transformation in tobacco and rice. The gusA reporter gene flanked by TM2 MAR in pBl121 and pCAMBIA-1301 vectors was controlled by a constitutive promoter or a photosynthetic tissue-specific promoter. The presence of TM2 MAR in different expression cassettes significantly increased the numbers of kanamycin-resistant tobacco shoots, hygromycin-resistant rice calli and shoots. Seeds from the independent transgenic lines with TM2 MAR can germinate normally on the medium containing 500 mg l -1 kanamycin, whereas none of the seeds from the transgenic lines without TM2 MAR survived. Furthermore, RNA gel blot analysis revealed that nptll messenger RNA levels are more abundant in the independent transgenic lines with TM2 MAR than in the lines without TM2 MAR. Altogether, these data reveal a possible mechanism that TM2 MAR improves the transformation frequency by increasing nptll gene expression.
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tm2 a novel strong Matrix Attachment Region isolated from tobacco increases transgene expression in transgenic rice calli and plants
Theoretical and Applied Genetics, 2005Co-Authors: Hua Xue, Guodong Yang, Yutao Yang, Mengmeng Zhang, Chengchao ZhengAbstract:Nuclear Matrix Attachment Regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear Matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.
Tianyun Wang - One of the best experts on this subject based on the ideXlab platform.
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distance effect characteristic of the Matrix Attachment Region increases recombinant protein expression in chinese hamster ovary cells
Biotechnology Letters, 2020Co-Authors: Jun He Zhang, Jihong Zhang, Xiaoyin Wang, Tianyun WangAbstract:Previously, we have found that the Matrix Attachment Region (MAR) may confer a ‘distance effect’ on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism. Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.
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novel short synthetic Matrix Attachment Region for enhancing transgenic expression in recombinant chinese hamster ovary cells
Journal of Cellular Biochemistry, 2019Co-Authors: Yanlong Jia, Xiao Guo, Xiaoyin Wang, Tianyun WangAbstract:Matrix Attachment Regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.
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Human genome-derived TOP1 Matrix Attachment Region enhances transgene expression in the transfected CHO cells.
Biotechnology letters, 2019Co-Authors: Yanlong Jia, Xiao Guo, Xi-cheng Wang, Tianyun WangAbstract:To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) Matrix Attachment Regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.
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a short synthetic chimeric sequence harboring Matrix Attachment Region psar2 increases transgene expression in chinese hamster ovary cells
Bioscience Biotechnology and Biochemistry, 2017Co-Authors: Wen Wang, Xiao Guo, Yanlong Jia, Yanfang Wang, Tianyun WangAbstract:A chimeric DNA fragment containing an interferon-beta Matrix Attachment Region (MAR) and an immunoglobulin MAR (PSAR2) was synthesized. PSAR2 was cloned into the upstream or downstream Region of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, which was then transfected into CHO cells. The results showed that PSAR2 did not effectively increase transgene expression when it was cloned into the upstream Region of the eGFP expression cassette. However, when inserted downstream of the eGFP expression cassette, PSAR2-enhanced transient transgene expression and significantly increased the numbers of stably transfected cells compared with the control vector. Additionally, PSAR2 significantly increased eGFP copy numbers as compared with the control vector. PSAR2 could significantly enhance transgene expression in CHO cells according to the position in the vector and increased transgene copy numbers. We found a short chimeric sequence harboring two MARs effectively increased ...
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Matrix Attachment Region combinations increase transgene expression in transfected chinese hamster ovary cells
Scientific Reports, 2017Co-Authors: Chunpeng Zhao, Jun He Zhang, Xiao Guo, Yanlong Jia, Sijia Chen, Yun Yang, Shaonan Chen, Tianyun WangAbstract:Matrix Attachment Regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.
Terumi Kohwishigematsu - One of the best experts on this subject based on the ideXlab platform.
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the Matrix Attachment Region binding protein satb1 interacts with multiple elements within the gp91phox promoter and is down regulated during myeloid differentiation
Journal of Biological Chemistry, 2001Co-Authors: Shannon M Hawkins, Terumi Kohwishigematsu, David G SkalnikAbstract:Abstract The gp91phox gene encodes a component of the respiratory burst NADPH oxidase complex and is highly expressed in mature myeloid cells. The transcriptional repressor CCAAT displacement protein binds to at least five sites within the proximal gp91phox promoter and represses expression prior to terminal phagocyte differentiation. The DNA binding activity of CCAAT displacement protein decreases during terminal phagocyte differentiation, thus permitting the binding of transcriptional activators and induction of gp91phox expression. We report here that the Matrix Attachment Region-binding protein SATB1 interacts with at least seven sites within the −1542 to +12-base pair gp91phox promoter. Four additional binding sites for CCAAT displacement protein were also identified. Furthermore, the most proximal SATB1-binding site within the gp91phox promoter binds specifically to the nuclear Matrix fraction in vitro. SATB1 expression is down-regulated during terminal myeloid cell differentiation, coincident with induction of gp91phoxexpression. Transient transfection assays demonstrate that a SATB1-binding site derived from the gp91phox promoter represses promoter activity in cells expressing SATB1. These findings underscore the importance of transcriptional repression in the regulation of gp91phox expression and reveal a candidate myeloid cell target gene for SATB1, a factor previously found to be essential for T cell development.
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a thymocyte factor satb1 suppresses transcription of stably integrated Matrix Attachment Region linked reporter genes
Biochemistry, 1997Co-Authors: Terumi Kohwishigematsu, Karin Maass, Jurgen BodeAbstract:SATB1 specifically recognizes and binds to specialized genomic Regions with an ATC sequence context with high base-unpairing propensity. Such base-unpairing Regions (BURs) are typically identified within nuclear scaffold- or Matrix-Attachment Regions (S/MARs). SATB1 is a homeodomain protein and is predominantly expressed in thymocytes. We obtained BHK cell lines expressing low levels of SATB1 by stable transfection and investigated its effect on stably integrated MAR-linked SV40 enhancer/promoter-driven luciferase reporter genes. For this study, both naturally occurring and synthetic MARs, as well as an AT-rich non-MAR control, were tested. Previous studies demonstrated that MAR sequences augment transcription of the linked reporter luciferase gene. Here, we show that SATB1 dramatically reduces the high levels of MAR-linked luciferase gene transcription. Transcription was virtually abolished for a reporter gene surrounded by two MARs at the 5‘ and 3‘ ends of the gene, which otherwise confer the highest le...
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an atypical homeodomain in satb1 promotes specific recognition of the key structural element in a Matrix Attachment Region
Journal of Biological Chemistry, 1997Co-Authors: Liliane A Dickinson, Craig D Dickinson, Terumi KohwishigematsuAbstract:Abstract SATB1 is a cell type-specific nuclear Matrix Attachment Region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing Region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3′ MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5′-(C/A)TAATA-3′ to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.
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contrasting effects of the satb1 core nuclear Matrix Attachment Region and flanking sequences of the keratin 18 gene in transgenic mice
Molecular Biology of the Cell, 1996Co-Authors: N Neznanov, Terumi Kohwishigematsu, R G OshimaAbstract:The 2.3 kb and 3.5 kb of DNA that flank the human keratin 18 (K18) gene and synthetic nuclear Matrix Attachment Regions (MAR) composed of the binding sites for the SATB1 nuclear protein were fused to a reporter gene that utilizes the mouse metallothionein promoter and the human growth hormone gene (MThGH). Transgenic mice were generated from both constructions and the control MThGH gene to test K18 and SATB1 MAR sequences for the ability to insulate the reporter gene from integration site-specific position effects. The MThGH control gene was variably expressed in brain, heart, intestine, kidney, liver, and testes, confirming previous studies. In contrast, the MThGH gene insulated by the K18 flanking sequences was expressed in the same tissues of four independent transgenic animals at levels correlated with the copy number except for intestine. The average level of expression on a per gene basis of the K18 insulated gene was from 9- to 49-fold higher than the control. The MThGH gene linked to the SATB1 MAR sequences was completely repressed in the brains and kidneys of all six transgenic mice. However, expression was nearly as efficient in testes as the K18-insulated gene. Both the SATB1 MAR and the K18 flanking sequences confer position-independent transcriptional status on the reporter gene in some or many tissues. However, the effects are stimulatory for the K18 elements and generally suppressive for the SATB1 MAR elements.
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a novel Matrix Attachment Region dna binding motif identified using a random phage peptide library
Journal of Biological Chemistry, 1995Co-Authors: Bingcheng Wang, Liliane A Dickinson, Erkki Koivunen, Erkki Ruoslahti, Terumi KohwishigematsuAbstract:SATB1 is a nuclear Matrix Attachment DNA (MAR)-binding protein which is predominantly expressed in thymocytes. This protein binds to the minor groove specifically recognizing an unusual DNA context exhibited by a specific MAR Region with strong base-unpairing propensity. A phage library displaying nonamer random peptides without any built-in structure was used to identify a MAR binding motif of SATB1. One predominant cyclic peptide C1 of CRQNWGLEGC selected by a MAR-affinity column showed 50% identity with a segment in SATB1 (amino acids 355-363). Replacement of the C1 similarity segment in SATB1 by a random amino acid sequence or its truncation resulted in more than 80% reduction in MAR binding. In contrast, replacement of the same SATB1 segment with the C1 peptide restored full MAR binding activity and specificity as the wild-type protein. Single amino acid mutation of the conserved Arg or Glu residue to Ala greatly reduced MAR binding. Taken together our data show that a nine amino acid sequence in SATB1 represents a key MAR binding motif. Phage display may provide a general tool for rapid identification of DNA binding peptide motifs.
R P Harbottle - One of the best experts on this subject based on the ideXlab platform.
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pepito a significantly improved non viral episomal expression vector for mammalian cells
BMC Biotechnology, 2010Co-Authors: Rudolf Haase, Suet Ping Wong, Orestis Argyros, R P Harbottle, Hans J Lipps, Manfred Ogris, Terese Magnusson, Maria Vizoso G Pinto, Juergen HaasAbstract:Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP) and directed into a 2000 bp long Matrix Attachment Region sequence (MARS) derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.
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persistent episomal transgene expression in liver following delivery of a scaffold Matrix Attachment Region containing non viral vector
Gene Therapy, 2008Co-Authors: Orestis Argyros, Andrew D Miller, Suet Ping Wong, Marcello Niceta, Simon N Waddington, Steven J Howe, Charles Coutelle, R P HarbottleAbstract:An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/Matrix Attachment Region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.
Steven Spiker - One of the best experts on this subject based on the ideXlab platform.
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correlations between scaffold Matrix Attachment Region s mar binding activity and dna duplex destabilization energy
Journal of Molecular Biology, 2006Co-Authors: Jurgen Bode, Ken Tsutsui, Steven Spiker, Silke Winkelmann, Sandra Gotze, A K Prashanth, Craig J BenhamAbstract:Scaffold or Matrix-Attachment Regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing Regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing Region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long Regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.
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the rb7 Matrix Attachment Region increases the likelihood and magnitude of transgene expression in tobacco cells a flow cytometric study
The Plant Cell, 2005Co-Authors: Christopher Halweg, William F Thompson, Steven SpikerAbstract:Many studies in both plant and animal systems have shown that Matrix Attachment Regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MARcontaining vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression.
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a tobacco Matrix Attachment Region reduces the loss of transgene expression in the progeny of transgenic tobacco plants
Plant Journal, 1999Co-Authors: Bekir Ulker, George Allen, William F Thompson, Steven Spiker, Arthur K WeissingerAbstract:Summary The RB7 Matrix Attachment Region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60-fold in stably transformed tobacco suspension cell lines. We have now used the same co-transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co-transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MARcontaining lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC 1 generation. Sixty-three per cent of the 21 MAR-containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology-dependent gene silencing.
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mfp1 a novel plant filament like protein with affinity for Matrix Attachment Region dna
The Plant Cell, 1996Co-Authors: Iris Meier, Steven Spiker, Tom Phelan, Wilhelm Gruissem, Dagmar SchneiderAbstract:The interaction of chromatin with the nuclear Matrix via Matrix Attachment Regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear Matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear Matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear Matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear Matrix and potentially with the nuclear envelope.